Letter
1038/nature17951
The industrial melanism mutation in British
peppered moths is a transposable element
Arjen E. van’t Hof1*, Pascal Campagne1*, Daniel J. Rigden1, Carl J. Yung1, Jessica Lingley1, Michael A. Quail2, Neil Hall1,
Alistair C. Darby1 & Ilik J. Saccheri1
Discovering the mutational events that fuel adaptation to
environmental change remains an important challenge for
evolutionary biology. The classroom example of a visible
evolutionary response is industrial melanism in the peppered
moth (Biston betularia): the replacement, during the Industrial
Revolution, of the common pale typica form by a previously
unknown black (carbonaria) form, driven by the interaction
between bird predation and coal pollution1. The carbonaria
locus has been coarsely localized to a 200-kilobase region, but the
specific identity and nature of the sequence difference controlling
the carbonaria–typica polymorphism, and the gene it influences,
are unknown2. Here we show that the mutation event giving rise
to industrial melanism in Britain was the insertion of a large,
tandemly repeated, transposable element into the first intron of
the gene cortex. Statistical inference based on the distribution of
recombined carbonaria haplotypes indicates that this transposition
event occurred around 1819, consistent with the historical record.
We have begun to dissect the mode of action of the carbonaria
transposable element by showing that it increases the abundance of
a cortex transcript, the protein product of which plays an important
role in cell-cycle regulation, during early wing disc development.
Our findings fill a substantial knowledge gap in the iconic example
of microevolutionary change, adding a further layer of insight into
the mechanism of adaptation in response to natural selection. The
discovery that the mutation itself is a transposable element will
stimulate further debate about the importance of ‘jumping genes’
as a source of major phenotypic novelty3.
Ecological genetics, the study of polymorphism and fitness in
natural populations, has been revitalised through the application of
next-generation sequencing technology to open up what were previ-
ously treated as genetic black boxes4,5. Growing appreciation of the
loci and developmental networks that generate adaptive phenotypic
variation6 promises to answer fundamental questions about the genetic
architecture of adaptation, such as the prevalence of genomic hotspots
for adaptation7, the relative contributions of major- and minor-effect
mutations8, and the structural nature and mode of action of benefi-
cial mutations9. Characterizing the identity and origin of functional
sequence polymorphisms provides the explicit link between the
mutation process and natural selection. In this context, while indus-
trial melanism in the peppered moth has retained its appeal as a
graphic example of the spread of a novel mutant rendered favour-
able by a major change in the environment, the crucial piece of the
puzzle that has been missing is the molecular identity of the causal
mutation(s)10.
A combined linkage and association mapping approach previously
localized the carbonaria locus to a <400-kb region orthologous to
Bombyx mori chromosome 17 (loci b–d)2. Thirteen genes and two
microRNAs occur within this interval, none of which was known to be
1
Institute of Integrative Biology, University of Liverpool, Biosciences Building, Crown Street, Liverpool L69 7ZB, UK. 2Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton,
Cambridgeshire CB10 1SA, UK.
*These authors contributed equally to this work.
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© 2016 Macmillan Publishers Limited. All rights reserved
doi:10.
involved in wing pattern development or melanization. By extending
the association mapping approach to a larger population sample and
more closely spaced genetic markers (see Methods), we narrowed the
carbonaria candidate region to about 100 kb (Fig. 1a). The candidate
region resides entirely within the span of one gene — the orthologue of
Drosophila cortex (cort), the only known function of which is as a
cell-cycle regulator during meiosis11. In B. betularia, cortex consists
of eight non-first exons, multiple alternative first exons (of which only
two, 1A and 1B, are strongly expressed in developing wing discs), and
a very large first intron (Fig. 1b).
The rapid spread of carbonaria gave rise to strong linkage disequi-
librium2, such that many sequence variants are associated with the
carbonaria phenotype. This poses a challenge for isolating the specific
causal variant(s). We reasoned that if the carbonaria mutation arose
on an ancestral typica haplotype2, the hitchhiking variants should in
principle also be present at some frequency within the typica popula-
tion, leaving the causal variants as the only ones unique to carbonaria.
High-quality contiguous reference sequences were assembled from
tiled bacterial artificial chromosome (BAC) and fosmid clones, result-
ing in one carbonaria and three different typica core haplotypes (see
Methods and Extended Data Fig. 1). Alignment of these sequences
(Supplementary Data 1) revealed 87 melanization candidate polymor-
phisms (Fig. 1b and Supplementary Table 1), concentrated within the
large first intron of cortex (69–91 kb, depending on haplotype). Eighty-
five candidates were eliminated using an increasing number of typica
individuals to exclude rare variants. A single nucleotide polymorphism
(carbonaria_candidate_25) was eventually excluded on the basis of one
individual out of 283 typica, leaving a very large insert (carbonaria_
candidate_45) as the only remaining candidate.
The insert was found to be present in 105 out of 110 fully black
moths (wild caught in the UK since 2002) and absent in all (283) typica
tested (see Methods and Extended Data Fig. 2). Consistent with local
carbonaria morph frequencies of 10–30% (ref. 12), 2 out of 105 indi-
viduals were homozygous for the carbonaria insert. Five individuals
that were morphologically indistinguishable from carbonaria did not
possess the carbonaria insert; they do not present any strong haplotype
association based on this set of candidate loci but do all differ from
the core carbonaria haplotype at many positions. Our interpretation is
that these individuals are hetero- or homozygous for the most extreme
of the insularia alleles (intermediate phenotypes), which are known
to occasionally produce carbonaria-like phenotypes13,14 and segre-
gate as alleles of the carbonaria locus in classical genetics crosses14.
Conversely, none of the genotyped insularia morphs (31 individuals,
covering the full spectrum of variation from i1 to i3 (ref. 14)) contains
the carbonaria insert (Extended Data Fig. 2). We conclude that the
large insert is the carbonaria mutation.
The carbonaria insert is 21,925 nucleotides long and is composed
of a roughly 9-kb essentially non-repetitive sequence (except for

b c
b
9 7 5 4 2
c
CCTCTGTA AC RU3 RU2
SRU7–9
CCT C
Figure 1 | The carbonaria candidate region, and the position and
structure of the carbonaria mutation. a, Approximately 400-kb candidate
region (bounded by marker loci b and d (ref. 2)) indicating gene content
and genotyping positions (vertical lines in the continuous grey bar).
Intron–exon structure and orientation are illustrated separately for each
gene (annotated in GenBank accession KT182637). b, Refined candidate
region including candidate polymorphisms (lines on the grey bar). The
intron–exon structure of cortex is shown for carbonaria (black moth)
and typica (speckled moth), highlighting the presence of a large (22 kb)
indel (orange) within the first intron. Exons 1A and 1B are alternative
transcription starts followed by the shared exons 2–9. c, The only exclusive
approximately 370 nucleotides at the repeat unit junctions) that is
tandemly repeated approximately two and one-third times, with only
minor differences among the repeats (Fig. 1c). The insert bears the
hallmark of a class II (DNA cut-and-paste) transposable element: short
inverted repeats (6 bp) and duplication of the (4-bp) target site present
in typica haplotypes (Extended Data Fig. 3). We estimate that there
are approximately 255 and 60 genomic copies, respectively, of the 9-kb
carbonaria transposable element (carb-TE) repeat unit and repeat unit
junctions, implying that there are relatively few genomic copies of
the complete carb-TE. No nucleotide or translated BLAST hits were
found in any relevant database, with the exception of B. betularia
RNA-sequencing (RNA-seq) reads (NCBI: SRX371328), indicating
that the carb-TE repeat unit is Biston-specific.
To examine patterns of recombination, which provide insight into
the evolutionary dynamics of a chromosomal region, we genotyped
the same 105 carbonaria and a sub-set of 37 typica, plus 35 insularia,
at 119 polymorphic loci within 28 PCR fragments distributed across
200 kb either side of the carb-TE (Fig. 1a). Diploid genotypes were
phased, and the resulting haplotypes divided into those with and with-
out the carb-TE. The sequence identity of the ancestral carbonaria
haplotype, whose core was known from the BAC and fosmid work,
was extended by assigning allelic state at each marker locus to ancestral
carbonaria or typica/insularia. Fifty per cent of carb-TE haplotypes had
retained the ancestral carbonaria haplotype across the full 400-kb win-
dow, and the remainder showed varying degrees of recombination with
typica haplotypes on one or both sides of the causal mutation (Fig. 2a).
The recent selective sweep15 is reflected by declining linkage disequi-
librium between the carbonaria locus and marker loci with increasing
genetic distance (Fig. 2b). The tenure of the carb-TE has been tran-
sient, having declined from ~99% to less than 5% in its industrial
heartland since 1970 (ref. 16). It has nevertheless left a substantial trace
of its former abundance in the form of ancestral carbonaria haplotype
blocks introgressed into typica and insularia haplotypes, consistent
with the simulation-based expectation (Fig. 2c).
© 2016 Macmillan Publishers Limited. All rights reserved
Letter RESEARCH
10 kb d
1B 1A
10 kb
RU1 GTTACACCTC
SRU4–6 SRU1–3
1 kb
carbonaria–typica polymorphism within the candidate region. The
structure of the insert, shown in the carbonaria sequence, corresponds to
a class II DNA transposon, with direct repeats resulting from target site
duplication (black nucleotides) next to inverted repeats (red nucleotides).
Typica haplotypes (lower sequence) lack the 4-base target site duplication,
the inverted repeats and the core insert sequence. The transposon consists
of ~9 kb tandemly repeated two and one-third times (repeat unit
(RU)1–RU3), with three short tandem subrepeat units (green dots,
SRU1–SRU9) within each repeat unit. Moth images were created from
photographs taken by A.E.v.H.
The first reported sighting of the carbonaria form is generally
regarded as having occurred in 1848 in Manchester1, although the
wording of the record implies that it was rare but not completely
unknown at this time. Establishing how long before this date the
carbonaria mutation occurred is complicated because it could
have existed undetected at a low frequency for hundreds of years
(Supplementary Methods). Our approach to this problem was to
infer the age of the mutation event independently by considering the
erosion of the ancestral carbonaria haplotype due to genetic recombi-
nation and mutation. One million simulated time trajectories of the
carbonaria phenotype were randomly drawn according to their fit to
historical frequency data (Extended Data Fig. 4). Based on these tra-
jectories, recombination patterns were simulated using an empirical
estimate of recombination rate and compared to the observed recom-
bination pattern of the carbonaria haplotypes. The probability density
for the date of the carb-TE mutation event (Fig. 2d) is highly skewed
(median, 1763; interquartile range, 1681–1806) with a maximum like-
lihood at 1819, a date highly consistent with a detectable frequency
being achieved in the mid-1840s.
The position of the carb-TE suggests that its effect on melanization
is achieved by altering the expression of cortex through one of several
potential mechanisms17 (incorporation of any part of the carb-TE
into cortex transcripts has been excluded). Biston cortex is charac-
terized by numerous splice isoforms and alternative first exons; we
focus on the population of transcripts initiated by exons 1A and 1B,
as the other first exons are absent or only weakly expressed in Biston
wing discs, and did not exhibit morph-specific differences (Extended
Data Fig. 5). The global pattern of splice isoforms showed neither con-
sistent presence or absence nor crude relative abundance differences
among morphs for any developmental stage (Extended Data Figs 6, 7).
Cumulative expression across all isoforms (Fig. 3a) increases by an
order of magnitude between the sixth larval instar (La6) and day 4
prepupa (Cr4), coinciding with a phase of rapid wing disc morphogen-
esis (Fig. 3b), and falls back to a low level by day 6 prepupa (Cr6) with
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 RESEARCH Letter

a b Figure 2 | Recombination pattern and ageing of

7 0.8 the carb-TE mutation. a, Nearest recombination
between carbonaria (carb-TE present (orange))
14
and non-carbonaria (typica and insularia (light
0.6 grey)) haplotypes (n = 107), 200 kb either side of
Number of haplotypes

32 the carb-TE (at position 0). Dark grey

areas indicate boundaries within which
0.4 recombination occurred. b, Multilocus linkage

rd
disequilibrium (rd) across the same sequence
window among carbonaria and non-carbonaria
0.2
54 haplotypes. Grey area indicates the widest 99%
confidence region, across loci, for the null
hypothesis (rd ≈ 0). Red lines represent the
0
simulation-based upper bound under the extreme
–200 –100 0 0 100 200 –200 –100 0 0 100 200 assumption that all alleles defining the carbonaria
Position (kb) Position (kb) haplotype were initially exclusive to it (mean and
c d 90% interval). c, Introgression of the ancestral
0.5 carbonaria haplotype (black) into non-carbonaria
haplotypes (grey; carb-TE absent (n = 144)). Red
Proportion of carbonaria haplotype

0.006 lines represent the simulation-based expectations

0.4
(mean and 90% interval). d, Probability density
Probability density

for the age of the carb-TE mutation inferred from

0.3 0.004 the recombination pattern in the carbonaria
haplotypes (maximum density at 1819 shown
0.2
by dotted line; first record of carbonaria in 1848
shown by dashed line).
0.002
0.1

0 0
–200 –100 0 0 100 200 1000 1200 1400 1600 1800
Position (kb) Year

no clear difference among morphs (t/t versus c/t, P > 0.5). To exclude ovaries11 (several cortex transcripts are expressed in B.betularia ovaries
interference by potentially non-functional isoforms, we targeted full and testes; Extended Data Fig. 5). The molecular function of cortex is
transcripts only, starting with either 1A or 1B. The abundance of the suggested by phylogenetic analysis, which indicates that Biston cortex
1B full transcript shows a consistent trend across several families with occurs in a lepidopteran sub-group within an insect-specific clade of
different genetic backgrounds (c/c > c/t > t/t) that is most pronounced a protein family containing the cell-cycle regulators cdc20 and cdh1,
at Cr4 (Fig. 3c and Extended Data Fig. 8a). The abundance of the encoded by fzy and rap (also known as fzr) in Drosophila (Extended
1A-initiated full transcript, which is in general an order of magnitude Data Fig. 9b). These proteins help to regulate fundamental cell division
less than that of the 1B transcript, does not show a significant differ- processes such as cytokinesis by presenting substrates to, and activating,
ence between genotypes (Fig. 3d and Extended Data Fig. 8b). the anaphase-promoting complex or cyclosome (APC/C), which ubiq-
The role of cortex in wing pattern melanization is not obvious. uitinates cell-cycle proteins, thereby earmarking them for degradation.
In Drosophila, cortex has been primarily associated with meiosis in Substrate recognition occurs by binding to degrons (short linear motifs

Figure 3 | Relative expression of cortex in

a b
0.020 developing wings of B. betularia. a, Average
expression (across typica and carbonaria morphs)
La6 Cr2 Cr3
of all cortex splice variants (exons 7–9) relative to
0.015
the control gene α-Spec in wing discs at different
Relative expression

developmental stages (La6, sixth instar larvae;

0.010 Cr2, day 2 crawler; Pu2, day 2 pupae; PDP, post-
Cr4 Cr5 Cr6 diapause pupae). Bars are s.e.m. b, Scaled images
0.005
(created from photographs taken by I.J.S.) of
B. betularia forewings at different stages.
c, d, Tukey plots for relative expression of cortex
0 2 mm 1B (c) and 1A (d) full transcripts in developing
La6 Cr2 Cr3 Cr4 Cr5 Cr6 Pu2 Pu4 Pu6 Pu8 PDP Pu4 PDP
wings of the three carbonaria-locus genotypes
c d (c/c, c/t and t/t) produced within the progeny of a
0.015 0.002 c/t × c/t cross (no data for c/c at Cr2). Genotypes
differ significantly for the 1B full transcript
Relative expression

(P < 0.001, generalized linear model (GLM)),

Relative expression

0.010 whereas genotypes do not differ for the 1A full

transcript (P > 0.2, GLM). (Note the differing
0.001
y-axes scales.) Equivalent graphs for the progeny
0.005 of c/t × t/t crosses (which lack the c/c genotype)
are presented in Extended Data Fig. 8.

0 0
cc ct tt cc ct tt cc ct tt cc ct tt cc ct tt cc ct tt cc ct tt cc ct tt cc ct tt cc ct tt
La6 Cr2 Cr3 Cr4 Cr5 La6 Cr2 Cr3 Cr4 Cr5

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© 2016 Macmillan Publishers Limited. All rights reserved

such as the D box and KEN box). Sequence conservation across lepi-
dopterans and non-lepidopterans reveals a single binding site in cortex
(Extended Data Fig. 9c) that probably binds the D box-like18 degron
LXEXXXN19. This degron binding capability is predicted for both of the
full isoforms (1A (441 amino acids) and 1B (407 amino acids), although
1B apparently lacks the N-terminal C box that is usually required for
APC/C binding) but not for the alternative isoforms (Extended Data
Table 1). These data demonstrate orthology and are consistent with
shared function of cortex between D. melanogaster and B. betularia,
although the molecular connection between cell-cycle protein degra-
dation at the APC/C and melanization remains to be determined.
Our results suggest that the carb-TE influences adult melanization
pattern by increasing the abundance of cortex, perhaps by altering the
course of scale-cell heterochrony, with dominance arising through a
threshold effect (the 1B full transcript is more abundant in c/c than
c/t). How the carb-TE promotes cortex expression is unknown but the
general mechanism is predicted to allow the production of insularia
morphs that are putatively controlled by different mutations within
cortex. In combination with parallel findings in Heliconius butterflies20,
our results support the idea that cortex is a conserved developmental
node for generating colour pattern variation in evolutionarily diverse
Lepidoptera. However, cortex may not be the only gene in this region
involved in patterning, as suggested by recent work on the B. mori
mutant Black moth, which has a similar phenotype to B. betularia
carbonaria21, although none of the genes implicated is differentially
expressed among carbonaria and typica wing discs.
The carb-TE is a spectacular example of an adaptively advantageous
transposon22–24; its discovery fills a fundamental gap in the peppered
moth story and furthers our appreciation of the mechanism under-
pinning rapid adaptation. A consensus on the general importance of
transposable elements for adaptive evolution has yet to emerge3,25.
Over longer time frames, phenotypic effects of transposable elements
may be obscured by imprecise excision that leaves a minimal trace
of the transposable element while retaining the mutant (adaptive)
phenotype26. By contrast, we have shown that the carb-TE is young,
approximately 200 years (generations) old, during which time it has
gone from a single mutation to near fixation (regionally) to near
extinction—driven by a pulse of environmental change.
Online Content Methods, along with any additional Extended Data display items and
Source Data, are available in the online version of the paper; references unique to
these sections appear only in the online paper.
received 3 July 2015; accepted 22 March 2016.
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Supplementary Information is available in the online version of the paper.
Acknowledgements The University of Liverpool Centre for Genomic Research
(M. Hughes, C. Bourne, R. Eccles, C. Hertz-Fowler and J. Kenny) performed
next-generation sequencing and Fragment Analyzer measurements. L. Cook
directed us to historical data sources. C. Bergman advised on transposon
detection. Population genetics simulations were performed on the University
of Liverpool Advanced Research Computing Condor service. This work was
supported by Natural Environment Research Council grants NE/H024352/1
and NE/J022993/1.
Author Contributions I.J.S., A.E.v.H. and P.C. designed the study and wrote the
paper; P.C., A.E.v.H. and D.J.R. produced the figures; A.E.v.H. directed molecular
biology experiments; A.E.v.H., C.J.Y. and J.L. conducted molecular biology
experiments; A.E.v.H. constructed the BAC and fosmid tilepaths; A.E.v.H. and
A.C.D. assembled, finished and annotated sequences; P.C. analysed population
genetic and gene expression data; I.J.S. collected the wild sample; I.J.S. and
C.J.Y. reared the samples and performed dissections; D.J.R. and A.E.v.H. built the
cortex tree; D.J.R. modelled the cortex structure; M.A.Q. constructed the fosmid
library; and A.C.D. and N.H. advised on the design of sequencing strategies.
Author Information The typica 1 haplotype (b–d interval) reference sequence
has been deposited in GenBank under accession number KT182637;
The B. betularia whole genome sequence has been deposited in the NCBI SRA
database under accession number SRX1060178; the cortex splice variants have
been deposited in GenBank under accession numbers KT235895–KT235906;
Rps3A has been deposited in GenBank under accession number JF811439;
α-spec has been deposited in GenBank under accession number KT182638.
Reprints and permissions information is available at www.nature.com/reprints.
The authors declare no competing financial interests. Readers are welcome to
comment on the online version of the paper. Correspondence and requests for
materials should be addressed to I.J.S. (saccheri@liverpool.ac.uk).
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 RESEARCH Letter
Methods
No formal statistical methods were used to predetermine sample sizes. The exper-
iments were not randomized, and investigators were not blinded to allocation
during experiments and outcome assessment.
Wild samples. Moths used for fine mapping and ageing analysis came from a
northwest England–north Wales transect sampled in 2002 (ref. 12), with 12 car-
bonaria and 6 insularia specimens additionally collected in 2005–2009.
Reference sequences. An extended BAC tiling path was constructed using mapped
B. betularia genes27, B. mori nscaf2829 (SilkDB) orthologues and BAC-end
sequences as probes. Combinatorial PCR using BAC-end sequences and internal
gene anchors were used to determine the relative positions of the BACs. Fosmids
were used to bridge gaps. A minimal tiling path was sequenced as a 3-kb mate-
pair library with Roche 454 GS FLX Titanium. Reads were assembled into contigs
using Newbler and manually scaffolded using tiled BAC-end sequences and exon
order of genes spanning multiple contigs as anchors. The scaffold covers a 3.6-Mb
region spanning the mapped genes Mhc to leucine-rich transmembrane protein
(lrtp) (GenBank accession numbers HM449891 and HM449887, respectively)
with the carbonaria polymorphism located towards the centre. A recombination
rate estimate within this region of 2.9 cM per Mb was obtained from a total of 350
offspring in 8 crosses screened for recombination between the ends of the 3.6-Mb
interval. Three typica and one carbonaria haplotype sequences were reconstructed
using BACs and fosmids for the region spanning locus b–d (Fig. 1a). Clones were
assigned to haplotypes on the basis of co-segregation of genotypes and phenotypes
between parents and sibs of the heterozygous (carbonaria–typica) individuals used
to generate the BAC (family 67) and fosmid (family 11) libraries. Small assem-
bly gaps caused by repetitive sections were bridged using long capillary Sanger
sequences; fosmid clone 25H14, containing the large repetitive transposable ele-
ment, was sequenced using Pacific Biosystems RS II to 300× coverage using P4-C2
chemistry and assembled using HGAP v2 (Pacific Biosystems). Homopolymer
length variation often caused by 454 errors rather than true polymorphisms was
verified by Sanger sequencing.
A draft genome assembly was generated from an individual homozygous for the
carbonaria region. Full-sib carbonaria–typica heterozygotes were crossed (family
135) to produce homozygous carbonaria offspring, as well as heterozygotes and
homozygous typica. The carbonaria homozygotes were identified using alleles
closely linked to the carbonaria locus, with more distant loci on either side used
to ensure that the haplotype had not been disrupted by recombination. DNA was
prepared by phenol–chloroform extraction from a final instar male larva with the
gut removed. The genome was sequenced to ~3.5 × coverage on a 454 FLX+ plat-
form and a draft assembly constructed using Newbler. The genome assembly was
used for polymorphism discovery, and for tiling path construction using homology
to B. mori. Single read coverage was used to detect repetitive regions, aiding in
single-target primer design, and to confirm the repetitive nature of the carb-TE.
The gene content of the b–d interval was examined by comparing its sequence
with GenBank proteins, expressed sequence tags (ESTs), transcriptomes, and
annotated genes in the orthologous region in other Lepidoptera. Tblastx against
these orthologous regions and Augustus28 gene prediction were used to detect
potentially overlooked genes. All genes were manually annotated and (except for
vcpl) confirmed using cDNA. The annotation of 11 genes (not including cortex)
was also subsequently confirmed against a B. betularia transcriptome (GenBank
SRX371328) assembled with Trinity29. MicroRNAs were found using miRBase with
blastn including hairpin precursors. BLAST (blastn, blastx) searches for carb-TE-
like sequences were performed on NCBI databases (GenBank nucleotide, protein,
EST, transcriptome), independently curated lepidopteran genome assemblies (for
example, SilkDB), and RepBase (19.09).
Fine mapping. The interval containing the carbonaria polymorphism was nar-
rowed down to a section bordered on both sides by evidence of carbonaria haplo-
type breakdown caused by recombination. Polymorphisms at regular intervals in
the b–d region (Fig. 1a; Supplementary Table 2) were genotyped in wild-caught
carbonaria, typica and insularia (105, 33 and 30 individuals, respectively). We
conservatively used only homozygous genotypes to set these boundaries because
the dominance of carbonaria obscures the assignment of alleles in heterozygous
genotypes to a certain morph haplotype. The four contiguous haplotype sequences
(one carbonaria and three typica) constructed from BACs and fosmids were aligned
between these narrowed-down boundaries and examined for polymorphisms that
were distinct in the carbonaria haplotype relative to all three typica haplotypes,
resulting in 87 carbonaria candidate polymorphisms (Extended Data Fig. 1 and
Supplementary Table 1). With the exception of carbonaria_candidate_45, wild-
caught typica were genotyped at all loci by means of PCR– restriction-fragment
length polymorphism (RFLP), PCR–indel or sequencing. Depending on the fre-
quency of the candidate alleles in the typica sample, 16 to 283 typica (32 to 566
typica haplotypes) were used for exclusion. Carbonaria_candidate_25 was present
© 2016 Macmillan Publishers Limited. All rights reserved
in only one out of 566 typica haplotypes. The typica phenotype of this individual
(12-2002-01) was confirmed, as was the presence of the carbonaria_candidate_25
allele from independently extracted DNA. A very large indel, later identified as
the true carbonaria polymorphism (carbonaria_candidate_45), that could not be
bridged by PCR required an alternative present/absent screening approach which
also provided a positive control for absence haplotypes (to distinguish insert
absence from PCR failure). A three-primer PCR was designed with two prim-
ers flanking the indel and a third within the insert, relatively close to the indel
boundary (Extended Data Fig. 2). The assay was validated using a family known
to include all three genotypes (family 135, Extended Data Fig. 2).
Inferring haplotypes and the age of the carbonaria mutation. A set of 177 indi-
viduals, including 105 carbonaria individuals, was genotyped at 119 polymorphic
loci within 28 PCR products, stretching across ~400 kb (Supplementary Table 2).
Carbonaria haplotypes were inferred using SHAPEIT30 and the position (interval)
of recombination breakpoints inferred based on two or more consecutive phase-
switched polymorphisms. High repeatability of the phasing outcomes was verified
by resampling, and switch errors were minimized by including known haplotypes
and classifying only two types (melanic and non-melanic). Indices of multilocus
linkage disequilibrium (rd) were calculated from polymorphisms within each PCR
fragment and the carbonaria locus across the 400-kb interval31. Their significance
was assessed using 999 Monte-Carlo permutations. The pattern of introgression of
the carbonaria haplotype into background haplotypes (that is, typica and insularia
morph alleles) was assessed using ChromoPainter v2 (ref. 32) to search for contig-
uous blocks that match the carbonaria haplotype, thus generating the ‘expectation
painting’ of background haplotypes.
The age of the carbonaria mutation was inferred with a simulation-based
approach. The analysis was performed in three steps. First, 1,000,000 time-
forward trajectories of the carbonaria phenotype were sampled, using a
Metropolis–Hastings algorithm, depending on their likelihood given historical
phenotypic frequencies (Supplementary Table 3), and conditional to their starting
date (x0) and population size (N). Second, recombination patterns were simulated
using the sampled trajectories, in populations of size N, and a fixed recombination
rate of 2.9 cM per Mb (males only). This process yielded sample distributions of
the closest recombination breakpoint relative to the carbonaria locus. Finally, the
likelihood of the simulated distributions given the empirical recombination pattern
was computed and averaged across simulations to estimate the probability density
of the mutation age (x0). For full details, see Supplementary Methods.
Code availability. Code available on request.
Expression and alternative transcripts of cortex. Offspring from either heterozy-
gous carbonaria/typica (c/t) × homozygous t/t crosses segregating 1:1 or c/t × c/t
crosses segregating 1 c/c: 2 c/t: 1 t/t were used for end-point reverse transcription
PCR (RT–PCR) and real-time quantitative PCR (qPCR) experiments. Caterpillars
were reared on grey willow (Salix cinerea). Wing discs (forewings and hindwings)
were dissected from final (sixth) instar larvae, crawlers or prepupae (days 2–6 from
the start of crawling stage), pre-diapause pupae (days 2–8 from pupation, at which
point they have entered diapause) and post-diapause pupae (wing discs staged
into six categories), and stored in RNAlater (Ambion). RNA was extracted with
TRIzol and cDNA synthesized with SuperScript III (Invitrogen)–oligo(dT). The
genotype–phenotype (adult morph) of each wing disc specimen was determined
with the carb-TE three primer PCR (and verified by sequencing a linked single
nucleotide polymorphism (SNP), carbonaria_candidate_25). Relative abundance
and qPCR data were analysed using generalized linear (mixed) models (GLM).
See Extended Data Fig. 8c for sample sizes.
Quantitative PCR experiments were designed to measure the relative abundance
of cortex transcripts, either of all transcripts combined (using primers in exons
7 and 9) or full transcripts only (primers in exons 1A-3 and 1B-3, as exon 3 is
effectively exclusive to the full transcripts (Extended Data Fig. 7)). DNase treatment
was not performed, but for exons 7–9 qPCR co-amplification of genomic DNA
was prevented by positioning the reverse primer on the exon 8–9 boundary (this
was not a concern for exons 1–3 qPCR because the large first intron precluded
genomic DNA amplification). We chose 40S ribosomal protein S3a (RpS3A)33 and
α-spec34 as two single-copy autosomal housekeeping genes. Primer sequences are
listed in Supplementary Table 4. Annealing temperatures were optimised to 66 °C
and amplicons were confirmed to produce single bands on agarose gels. cDNA was
diluted 1:1 with water to allow template volumes within the accuracy range of the
pipette used. Quantitative PCRs for target and control were run in three replicates
using Kapa SYBR Fast qPCR Universal under recommended conditions on a Roche
LightCycler 480 with 45 cycles and a melting curve. As both control genes gave
similar results, only α-spec was used for the entire sample.
Alternative transcription starts of cortex were searched for using 5′ rapid
amplification of cDNA ends (RACE) on RNA extracted from 15 wing disc sam-
ples covering a wide range of stages and c/c, c/t and t/t genotypes, and also from

whole pupae and testes. Cortex-specific cDNA was synthesized with SuperScript
III and a gene-specific negative strand primer; 5′ cytosine extension was added
using terminal transferase (NEB) and deoxycytidine triphosphates (dCTPs). The
single-stranded cDNA was made double-stranded and a target sequence for ampli-
fication incorporated in a single extension cycle (LongAmp Hot Start, NEB) with
an oligonucleotide containing a 5′ primer recognition site and a 3′ poly-G tail. PCR
was performed using a forward primer matching the synthetic 5′ end and a nested
cortex-specific reverse primer. The amplicons were sequenced using a second
nested primer. The alternative first exons were confirmed by Sanger sequencing
with forward primers inside the newfound exons to generate clean sequence with-
out the background noise commonly observed with 5′ RACE.
The complete pattern of cortex splice variation was examined with end-point
RT–PCR using primers Bb_cort_exon1A_F or Bb_cort_exon1B_F and Bb_cort_
exon9_R (for primer sequences see Supplementary Table 4). PCR conditions
were 60 °C annealing, 40 cycles, 75 s extension, 25 μl total volume, 3 μl wing disc
cDNA, LongAmp Taq DNA polymerase (NEB). A Fragment Analyzer (Advanced
Analytical) was used to estimate the size and relative abundance of amplicons
within each individual, after normalizing samples to a concentration range of
~1–10 ng μl−1. The concentration of each fragment peak was calculated using
PROSize (Advanced Analytical), and the relative abundance was computed as the
concentration of a splice variant divided by the sum of all fragment concentrations
within that individual profile. The cortex splice variant amplicons were sequenced
as two pools (t/t and c/t) using Pacific Biosystems RS II with P6-C4 chemistry and
the insert reads extracted using smrtportal (Pacific Biosystems). Reads that con-
tained exon 1A or 1B and exon 9 were used to validate the sequence composition
and relative abundance of spliced gene isoforms.
No part of carb-TE was detected in cortex transcripts, either with PacBio
sequencing or with PCR using various primer combinations where one primer
lies within the transposon and the other matches a cortex exon. However, a carb-
TE-like partial sequence was amplified (with primers within repeat units) from
both typica and carbonaria morph cDNA synthesized using carb-TE primers,
implying that these RNA sequences are transcribed from non-allelic homologues
of the carb-TE.
Expression of alternative candidate genes. Two B. mori adult melanism/pattern-
ing mutations, Black moth (Bm) and Wild wing spot (Ws), were recently mapped to
a region partially orthologous to the carbonaria interval21. In this study, end-point
PCR showed complete absence of cortex expression in pupal stages and adults but
potentially important prepupal stages were not examined. Three neighbouring
genes (BGIBMGA005658, BGIBMGA005657 and BGIBMGA005655) did show
convincing differences between the wild type and both mutants even though these
genes lie outside the Ws mapping interval. We performed equivalent end-point
RT–PCR for the three orthologues in B. betularia to determine whether morph–
gene expression associations existed between carbonaria and typica (comparing
c/t and t/t genotypes for wing disc stages Cr4, Cr6, Pu2, Pu4 and PDP). PCR
© 2016 Macmillan Publishers Limited. All rights reserved
Letter RESEARCH
conditions were as for cortex 1A/1B–9 end-point PCRs, except for 45-s extension
(for primer sequences, see Supplementary Table 4).
Cortex phylogeny and protein modelling. Cortex sequences derived from data-
base searches (Supplementary Table 5) were supplemented with a selection of
chd1 and cdc20/ fzy sequences from model organisms and the set aligned with
MAFFT35 (Supplementary Data 2). The central propeller domain was isolated and
used for bootstrapped phylogenetic analysis with MEGA 6 (ref. 36) employing its
Maximum Likelihood algorithm and the JTT matrix-based model. Any gapped
positions were ignored. Homology models of B. betularia and D. melanogaster
cortex proteins were made with MODELLER37 and Consurf38 was used to map
protein sequence conservation to their respective surfaces among lepidopteran or
non-lepidopteran cortex proteins.
27. van’t Hof, A. E. et al. Linkage map of the peppered moth, Biston betularia
(Lepidoptera, Geometridae): a model of industrial melanism. Heredity 110,
283–295 (2013).
28. Stanke, M. & Morgenstern, B. AUGUSTUS: a web server for gene prediction in
eukaryotes that allows user-defined constraints. Nucleic Acids Res. 33,
W465–W467 (2005).
29. Grabherr, M. G. et al. Full-length transcriptome assembly from RNA-Seq data
without a reference genome. Nature Biotechnol. 29, 644–652 (2011).
30. Delaneau, O., Marchini, J. & Zagury, J.-F. A linear complexity phasing method
for thousands of genomes. Nature Methods 9, 179–181 (2011).
31. Agapow, P.-M. & Burt, A. Indices of multilocus linkage disequilibrium. Mol. Ecol.
Notes 1, 101–102 (2001).
32. Lawson, D. J., Hellenthal, G., Myers, S. & Falush, D. Inference of population
structure using dense haplotype data. PLoS Genet. 8, e1002453 (2012).
33. Baxter, S. W. et al. Genomic hotspots for adaptation: the population genetics of
Müllerian mimicry in the Heliconius melpomene clade. PLoS Genet. 6,
e1000794 (2010).
34. Reed, R. D., McMillan, W. O. & Nagy, L. M. Gene expression underlying adaptive
variation in Heliconius wing patterns: non-modular regulation of overlapping
cinnabar and vermilion prepatterns. Proc. R. Soc. Lond. B 275, 37–45 (2008).
35. Katoh, K. & Standley, D. M. MAFFT multiple sequence alignment software
version 7: improvements in performance and usability. Mol. Biol. Evol. 30,
772–780 (2013).
36. Tamura, K., Stecher, G., Peterson, D., Filipski, A. & Kumar, S. MEGA6: molecular
evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30, 2725–2729
(2013).
37. Šali, A. & Blundell, T. L. Comparative protein modelling by satisfaction of spatial
restraints. J. Mol. Biol. 234, 779–815 (1993).
38. Ashkenazy, H., Erez, E., Martz, E., Pupko, T. & Ben-Tal, N. ConSurf 2010:
calculating evolutionary conservation in sequence and structure of proteins
and nucleic acids. Nucleic Acids Res. 38, W529–W533 (2010).
39. Muñoz-López, M. & García-Pérez, J. L. DNA transposons: nature and
applications in genomics. Curr. Genomics 11, 115–128 (2010).
40. Chang, L., Zhang, Z., Yang, J., McLaughlin, S. H. & Barford, D. Atomic structure
of the APC/C and its mechanism of protein ubiquitination. Nature 522,
450–454 (2015).
RESEARCH Letter
Extended Data Figure 1 | BAC and fosmid haplotype tilepaths used
to define carbonaria candidate polymorphisms. a, BAC and fosmid
tilepaths of the carbonaria haplotype (black bars) and three typica
haplotypes (different shades of grey). Two small regions not covered by
BACs or fosmids were reconstructed using parent and offspring sequences
from the same heterozygous family (FAM11). The positions of loci b and d
(see Figure 1) are indicated by the dashed lines, and the carbonaria
candidate region is highlighted blue. Fosmid 25H14 containing carb-TE
© 2016 Macmillan Publishers Limited. All rights reserved
appears small because it is aligned against the typica reference sequence,
which does not include the carb-TE. b, Alignment of three typica
haplotypes against the carbonaria haplotype for a short section within the
carbonaria candidate region, showing SNPs (dots are nucleotides identical
to the carbonaria sequence). Polymorphisms in which all three typica
alleles differed from carbonaria were treated as carbonaria candidates;
polymorphisms in which the same allele occurred in carbonaria and at
least one typica were excluded from further consideration.

Extended Data Figure 2 | Validation of the 3-primer PCR carb-TE
genotyping assay in a family and its application in a variety of wild-
caught moths. a, Schematic alignment of carbonaria and typica haplotypes
showing the position of the three primers (A, B and C, not to scale) used
in the same PCR to detect the presence and absence of the 22 kb carb-TE.
In the presence of the carb-TE, primers A and C are too far apart to
generate a product; the repeat structure of the carb-TE presents three
annealing sites for primer B but only the shortest primer B–C combination
is amplified when using 45-s extension (primer sequences are listed in
Supplementary Table 1). b, carb-TE genotypes for father (lane 2), mother
(lane 3) and 15 offspring (lanes 4–18); the two brightest bands in the size
ladder are 300 bp and 1 kb (lane 1). The parents were full siblings and
© 2016 Macmillan Publishers Limited. All rights reserved
Letter RESEARCH
known to be heterozygous (c/t), and therefore expected to generate c/c, c/t
and t/t offspring. The larger band (primers B–C) indicates the presence of
the carb-TE and the smaller band (primers A–C) its absence (typica allele
in this family); heterozygotes have both bands. The individual in lane 15
(135F1-12) is the homozygous male used for whole genome sequencing.
c, Presence or absence of the carb-TE in a carbonaria haplotype fosmid
clone (lane 2), three different typica haplotype clones (lanes 3–5; one
fosmid, two BACs), wild carbonaria homozygotes (lanes 6 and 7), wild
carbonaria heterozygotes (lanes 8–10), typica with a flanking haplotype
similar to the carbonaria haplotype but lacking the carb-TE (lanes 11–13),
light insularia (lanes 14–16), intermediate insularia (lanes 17–19), dark
insularia (lanes 20–22) and carbonaria-like insularia (lanes 23–25).
RESEARCH Letter

Extended Data Figure 3 | Hypothetical reconstruction of the birth
of the carbonaria allele. Class II non-autonomous DNA transposition
is mediated by two transposase monomers linked to terminal inverted
repeats (TIR). The monomers form a dimer at the target site that is
cleaved to leave short direct repeated overhangs. The transposable element
including TIRs is inserted and finally the single-stranded cleaved sites are
filled in to complete the target site duplication39. The unduplicated target
site motif (CCTC) is common, possibly ubiquitous, in all non-carbonaria
(typica and insularia) haplotypes, but a typica ancestor is more likely given
the pattern of haplotype similarities and the presumed prevalence of typica
haplotypes around 1800.

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 Letter RESEARCH

Extended Data Figure 4 | The rise and fall of carbonaria in the
Manchester area. a, Frequency of the carbonaria phenotype from ~1800 to
2009. b, Corresponding frequencies of the carbonaria allele. The envelopes
show the confidence intervals (50%, 90% and 99%) for the simulated
trajectories. Dark-red dots, observations falling within the simulated
trajectories; orange dots, additional data collected after 2002 (year during
which >85% of the field sample was collected). Stars indicate likely
frequencies where historical data are scarce. Data and sources are listed in
Supplementary Table 3.

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 RESEARCH Letter
Extended Data Figure 5 | Position and tissue-specific expression of
alternative first exons of the gene cortex. a, Illustration of cortex exon
structure indicating the positions of thirteen alternative transcription
starts and subsequent exons relative to the flanking genes in the b–d
region (position of carb-TE indicated by orange bar). b, Expression of
different starting position cortex transcripts. End-point RT–PCR with
reduced cycles (35) was used to exclude transcripts with negligible
dosage. Amplicon intensities are scaled between + (faint but visible) and
© 2016 Macmillan Publishers Limited. All rights reserved
+++(strong PCR product). Negative PCRs represent expression below
the detection threshold; this may even occur in ‘origin’ tissue types (wing
disc/pupa/testes) in which the alternative starts were discovered owing to
the fact that 5′ RACE used ~20 times the amount of RNA template relative
to the standard cDNA synthesis for the 35 cycle end-point PCRs. Ovaries
were not used for 5′ RACE, which may have caused gonad expression bias
towards testes. Test tissues are sixth instar larvae gonads and wing discs at
different developmental stages (abbreviations as in Fig. 3).
 Letter RESEARCH

Extended Data Figure 6 | Examples of cortex splice variation pattern in in the size ladder are 300 bp and 1 kb) and carbonaria individuals (all c/t
typica and carbonaria developing wing discs. End-point PCR on wing heterozygotes) to the right of the central ladder. a, Exon 1A variants in Cr2
disc cDNA amplified with primers in the first and last exons (E1–E9), with stage. b, Exon 1B variants in Cr4 stage. (See Fig. 3 for stage abbreviations.)
typica individuals to the left of the central ladder (the two brightest bands

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RESEARCH Letter
Extended Data Figure 7 | Exonic structure and size distributions of
cortex splice variants amplified by end-point RT–PCR with primers
in exon 1A or 1B and exon 9. Size distributions of the PacBio reads are
displayed for the two alternative first exons 1A (a) and 1B (b) of cortex.
c, d, Comparison of carbonaria locus genotypes (t/t pale blue fill, c/t light
blue line, c/c dark blue line) measured with Fragment Analyzer. Relative
fluorescence units (RFU) were averaged across individuals for fragments
amplified with E1A-E9 (c) or E1B-E9 (d) primers. Prior to averaging,
RFUs were standardized so that the total fluorescence (area under the
© 2016 Macmillan Publishers Limited. All rights reserved
curve) per individual scaled to 1. Arrows with the same numbers denote
either similar exonic structure (E1A versus E1B variants) or fragment
identity between the two sources of data (PacBio reads and Fragment
Analyzer). Exonic structure of the six main splice variants is represented
in matrices (a, b), in which white cells represent skipped exons in a splice
variant (asterisk indicates full transcript in which the first 71 bp of exon 6
are missing). Apparent differences among melanic and non-melanic for
1A number 2 and number 3 splice variants were not consistent among
families.
 Letter RESEARCH

Extended Data Figure 8 | Tukey plots for relative expression of cortex (P = 0.001, GLM), whereas genotypes do not differ for 1A full transcript
full transcript in developing wing discs. c/t heterozygotes are compared (P > 0.5, GLM). Note the differing y-axes scales. c, Sample sizes for cortex
with t/t homozygotes produced from c/t × t/t crosses (starting with exon qPCR experiments by wing disc developmental stage and carbonaria-locus
1B (a) or exon 1A (b)). Genotypes differ significantly for 1B full transcript genotype.

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 RESEARCH Letter
Extended Data Figure 9 | Orthology and functional domain conservation
of cortex protein. a, Schematic illustration, not to scale, of molecular
features of B. betularia cortex protein sequence. b, Bootstrapped Maximum
Likelihood consensus tree calculated with MEGA 6 of fzy/cortex derived
from the propeller domain of the alignment in Supplementary Data 2.
Branches are collapsed where partitions were reproduced in less than half
of bootstrap replicates. Major groups containing lepidopteran cortex (black
circles), non-lepidopteran cortex (red circles), fzr/rap (yellow circles) or
fzy/cdc20/cdh1 proteins (green circles) are similarly unequivocally defined in
trees obtained by neighbour joining or maximum parsimony methods (not
shown). c, 3D protein sequence conservation mapping of lepidopteran cortex
sequences onto a homology model of B. betularia cortex (top); all cortex
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sequences onto the same B. betularia model (middle); non-lepidopteran
cortex sequences onto a model of D. melanogaster cortex (bottom). Molecular
surfaces are shown in PyMOL using a spectrum from high (blue) to low (red)
conservation. The mapping reveals the shared presence of a presumed inter-
blade D box-like degron-binding site (pink segment is superimposed D box-
mimicking sequence from the structures of human APC/C (PDB accession
4ui9)40). In contrast, there is much weaker conservation of surface regions
corresponding to facial KEN box or helical specificity determinant sites
(white and grey ribbons, respectively, from the same structure), suggesting
that cortex proteins lack these functionalities. Note that the greater sequence
variability in the non-lepidopteran set leads to lower overall sequence
conservation (bottom) but that overall patterns in all panels are similar.
 Letter RESEARCH

Extended Data Table 1 | Predicted functionality of B. betularia cortex isoforms (starting with exon 1A or 1B)

*Isoforms as defined in Extended Data Fig. 7.

†As the region lost from the propeller fold constitutes approximately a single blade, it is possible that these, and only these, truncated-propeller forms may still fold stably.

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